Differences in TRPC3 and TRPC6 channels assembly in mesenteric vascular smooth muscle cells in essential hypertension

Producción CientíficaIncreased vascular tone in essential hypertension involves a sustained rise in total peripheral resistance. A model has been proposed in which the combination of membrane depolarization and higher L‐type Ca2+ channel activity generates augmented Ca2+ influx into vascular smooth muscle cells (VSMCs), contraction and vasoconstriction. The search for culprit ion channels responsible for membrane depolarization has provided several candidates, including members of the canonical transient receptor potential (TRPC) family. TRPC3 and TRPC6 are diacylglycerol‐activated, non‐selective cationic channels contributing to stretch‐ or agonist‐induced depolarization. Conflicting information exists regarding changes in TRPC3/TRPC6 functional expression in hypertension. However, although TRPC3‐TRPC6 channels can heteromultimerize, the possibility that differences in their association pattern may change their functional contribution to vascular tone is largely unexplored. We probe this hypothesis using a model of essential hypertension (BPH mice; blood pressure high) and its normotensive control (BPN mice; blood pressure normal). First, non‐selective cationic currents through homo‐ and heterotetramers recorded from transfected Chinese hamster ovary cells indicated that TRPC currents were sensitive to the selective antagonist Pyr10 only when TRPC6 was present, whereas intracellular anti‐TRPC3 antibody selectively blocked TRPC3‐mediated currents. In mesenteric VSMCs, basal and agonist‐induced currents were more sensitive to Pyr3 and Pyr10 in BPN cells. Consistently, myography studies showed a larger Pyr3/10‐induced vasodilatation in BPN mesenteric arteries. mRNA and protein expression data supported changes in TRPC3 and TRPC6 proportions and assembly, with a higher TRPC3 channel contribution in BPH VSMCs that could favour cell depolarization. These differences in functional and pharmacological properties of TRPC3 and TRPC6 channels, depending on their assembly, could represent novel therapeutical opportunities.Instituto de Salud Carlos III (project RD12/0042/0006)Programa Estatal de Investigación (project BFU2013-45867-R

High blood pressure associates with the remodelling of inward rectifier K+ channels in mice mesenteric vascular smooth muscle cells

Producción CientíficaThe increased vascular tone that defines essential hypertension is associated with depolarization of vascular smooth muscle cells (VSMCs) and involves a change in the expression profile of ion channels promoting arterial contraction. As a major regulator of VSMC resting membrane potential (VM), K+channel activity is an important determinant of vascular tone and vessel diameter. However, hypertension-associated changes in the expression and/or modulation of K+channels are poorly defined, due to their large molecular diversity and their bed-specific pattern of expression. Moreover, the impact of these changes on the integrated vessel functionand their contribution to the development of altered vascular tone under physiological conditions need to be confirmed. Hypertensive (BPH) and normotensive (BPN) mice strains obtained by phenotypic selection were used to explore whether changes in the functional expression of VSMC inward rectifier K+channels contribute to the more depolarized resting VM and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifierK+channel mRNA in several vascular beds from BPN and BPH animals, and their functional contribution to VSMC excitability and vascular tone in mesenteric arteries. We found a decrease in the expression of Kir2.1, Kir4.1, Kir6.x and SUR2 mRNA in BPH VSMCs, and a decreased functional contribution of both KIRand KATP channels in isolated BPH VSMCs. However, only the effect of KATP channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of KATP channels may be an important element in the remodelling of VSMCs in essential hypertension.Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grant R006/009)Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898)Junta de Castilla y León (grant VA094A11-2

Characterization of Ion Channels Involved in the Proliferative Response of Femoral Artery Smooth Muscle Cells

31 páginas, 6 figuras, adicionales 6 figuras y 3 tablas.[Objective]: Vascular smooth muscle cells (VSMCs) contribute significantly to occlusive vascular diseases by virtue of their ability to switch to a noncontractile, migratory, and proliferating phenotype. Although the participation of ion channels in this phenotypic modulation (PM) has been described previously, changes in their expression are poorly defined because of their large molecular diversity. We obtained a global portrait of ion channel expression in contractile versus proliferating mouse femoral artery VSMCs, and explored the functional contribution to the PM of the most relevant changes that we observed. [Methods and Results]: High-throughput real-time polymerase chain reaction of 87 ion channel genes was performed in 2 experimental paradigms: an in vivo model of endoluminal lesion and an in vitro model of cultured VSMCs obtained from explants. mRNA expression changes showed a good correlation between the 2 proliferative models, with only 2 genes, Kv1.3 and Kvβ2, increasing their expression on proliferation. The functional characterization demonstrates that Kv1.3 currents increased in proliferating VSMC and that their selective blockade inhibits migration and proliferation. [Conclusion]: These findings establish the involvement of Kv1.3 channels in the PM of VSMCs, providing a new therapeutical target for the treatment of intimal hyperplasia.This work was supported by Ministerio de Sanidad, Instituto de Salud Carlos III grants R006/009 (Red Heracles), FS041139-0 (M.R.), and PI041044 (J.R.L.-L.); Ministerio de Educacio´n y Ciencia grants BFU2004-05551 (M.T.P.-G.) and BFU2007-61524 (J.R.L.-L.); and Junta de Castilla y Leon grant GR242. Dr Moreno-Domínguez is a fellow of the Spanish Ministerio de Educacion y Ciencia.Peer reviewe

Dynamic changes of the extracellular matrix during corneal wound healing

Producción CientíficaThe extracellular matrix (ECM) confers transparency to the cornea because of the precise organization of collagen fibrils and a wide variety of proteoglycans. We monitored the corneal wound healing process after alkali burns in rabbits. We analyzed the location and expression of collagens and proteoglycans, the clinical impact, and the recovery of optical transparency. After the animals received both general and ocular topical anesthesia, the central cornea of the left eye was burned by placing an 8-mm diameter filter paper soaked in 0.5 N NaOH for 60 s. The eyes were evaluated under a surgical microscope at 1, 3, and 6 months after burning. At each time point, the clinical conditions of the burned and control corneas were observed. The arrangement of collagen fibers in the corneal stroma was visualized by Picrosirius-red staining, Gomori's silver impregnation and transmission electronic microscopy. Corneal light transmittance was also measured. Myofibroblasts presence was analyzed by immunohistochemistry. mRNA expression levels of collagen types I and III, lumican, decorin, keratocan and alpha-smooth muscle actin were determined by quantitative real-time polymerase chain reaction. One month after alkali burn, the ECM was disorganized and filled with lacunae containing different types of cells and collagen type III fibers in the wound area. Corneal opacities were present with attendant loss of light transmittance. Collagen and proteoglycan mRNA expression levels were up-regulated. After three months, wound healing progress was indicated by reduced corneal opacity, increased light transmittance, reorganization of collagen fibers and only collagen type I expression levels were at control levels. After six months, the wound area ECM morphology was similar to controls, but transmittance values remained low, denoting incomplete restoration of the stromal architecture. This multidisciplinary study of the stromal wound healing process revealed changes in corneal transmittance, collagen organization, myofibroblasts presence and ECM composition at 1, 3, and 6 months after alkali burning. Documenting wound resolution during the six-month period provided reliable information that can be used to test new therapies.Instituto de Salud Carlos III (Health Research Fund PI15/01906 )Ministerio de Economía, Industria y Competitividad ( project BFU2016-75360-R)Junta de Castilla y León (Project VA114P17

Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization

Producción CientíficaThe dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA. In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells.The Swedish Research Council (grant 2012-2197)The Crafoord Foundation (grant 20150629)Instituto de Salud Carlos III (grant RD12/0042/0006)Ministerio de Economía, Industria y Competitividad (grant BFU2013-45867-R

Contribution of Kv channels to phenotypic remodeling of human uterine artery smooth muscle cells

Producción CientíficaVascular smooth muscle cells (VSMCs) perform diverse functions that can be classified into contractile and synthetic (or proliferating). All of these functions can be fulfilled by the same cell because of its capacity of phenotypic modulation in response to environmental changes. The resting membrane potential is a key determinant for both contractile and proliferating functions. Here, we have explored the expression of voltage-dependent K+ (Kv) channels in contractile (freshly dissociated) and proliferating (cultured) VSMCs obtained from human uterine arteries to establish their contribution to the functional properties of the cells and their possible participation in the phenotypic switch. We have studied the expression pattern (both at the mRNA and at the protein level) of Kvα subunits in both preparations as well as their functional contribution to the K+ currents of VSMCs. Our results indicate that phenotypic remodeling associates with a change in the expression and distribution of Kv channels. Whereas Kv currents in contractile VSMCs are mainly performed by Kv1 channels, Kv3.4 is the principal contributor to K+ currents in cultured VSMCs. Furthermore, selective blockade of Kv3.4 channels resulted in a reduced proliferation rate, suggesting a link between Kv channels expression and phenotypic remodeling.Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009. FS041139-0 and PI041044)Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524)Junta de Castilla y León (grant GR242

Cell cycle-dependent expression of Kv3.4 channels modulates proliferation of human uterine artery smooth muscle cells

Producción CientíficaAims: Vascular smooth muscle cell (VSMC) proliferation is involved in cardiovascular pathologies associated with unwanted arterial wall remodelling. Coordinated changes in the expression of several K+ channels have been found to be important elements in the phenotypic switch of VSMCs towards proliferation. We have previously demonstrated the association of functional expression of Kv3.4 channels with proliferation of human uterine VSMCs. Here, we sought to gain deeper insight on the relationship between Kv3.4 channels and cell cycle progression in this preparation. Methods and results: Expression and function of Kv3.4 channels along the cell cycle was explored in uterine VSMCs synchronized at different checkpoints, combining real-time PCR, western blotting, and electrophysiological techniques. Flow cytometry, Ki67 expression and BrdU incorporation techniques allowed us to explore the effects of Kv3.4 channels blockade on cell cycle distribution. We found cyclic changes in Kv3.4 and MiRP2 mRNA and protein expression along the cell cycle. Functional studies showed that Kv3.4 current amplitude and Kv3.4 channels contribution to cell excitability increased in proliferating cells. Finally, both Kv3.4 blockers and Kv3.4 knockdown with siRNA reduced the proportion of proliferating VSMCs. Conclusion: Our data indicate that Kv3.4 channels exert a permissive role in the cell cycle progression of proliferating uterine VSMCs, as their blockade induces cell cycle arrest after G2/M phase completion. The modulation of resting membrane potential (VM) by Kv3.4 channels in proliferating VSMCs suggests that their role in cell cycle progression could be at least in part mediated by their contribution to the hyperpolarizing signal needed to progress through the G1 phase.Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009 and PI041044)Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524)Junta de Castilla y León (grant GR242

Characterization of ion channels involved in the proliferative response of femoral artery smooth muscle cells

Producción CientíficaObjective: Vascular smooth muscle cells (VSMCs) contribute significantly to occlusive vascular diseases by virtue of their ability to switch to a noncontractile, migratory, and proliferating phenotype. Although the participation of ion channels in this phenotypic modulation (PM) has been described previously, changes in their expression are poorly defined because of their large molecular diversity. We obtained a global portrait of ion channel expression in contractile versus proliferating mouse femoral artery VSMCs, and explored the functional contribution to the PM of the most relevant changes that we observed. Methods and Results: High-throughput real-time polymerase chain reaction of 87 ion channel genes was performed in 2 experimental paradigms: an in vivo model of endoluminal lesion and an in vitro model of cultured VSMCs obtained from explants. mRNA expression changes showed a good correlation between the 2 proliferative models, with only 2 genes, Kv1.3 and Kvβ2, increasing their expression on proliferation. The functional characterization demonstrates that Kv1.3 currents increased in proliferating VSMC and that their selective blockade inhibits migration and proliferation. Conclusion: These findings establish the involvement of Kv1.3 channels in the PM of VSMCs, providing a new therapeutical target for the treatment of intimal hyperplasia.Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009, FS041139-0 and PI041044)Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524)Junta de Castilla y León (grant GR242

K+ channels expression in hypertension after arterial injury, and effect of selective Kv1.3 blockade with PAP-1 on intimal hyperplasia formation

Producción CientíficaK+ channels are central to vascular pathophysiology. Previous results demonstrated that phenotypic modulation associates with a change in Kv1.3 to Kv1.5 expression, and that Kv1.3 blockade inhibits proliferation of VSMCs cultures. Purpose: To explore whether the Kv1.3 to Kv1.5 switch could be a marker of the increased risk of intimal hyperplasia in essential hypertension and whether systemic treatment with Kv1.3 blockers can prevent intimal hyperplasia after endoluminal lesion . Methods: Morphometric and immunohistochemical analysis were performed in arterial segments following arterial injury and constant infusion of the Kv1.3 blocker PAP-1 during 28 days. Differential expression of K+ channel genes was studied in VSMC from hypertensive (BPH) and normotensive (BPN) mice, both in control and after endoluminal lesion. Finally, the migration and proliferation rate of BPN and BPH VSMCs was explored in vitro. Results: Changes in mRNA expression led to an increased Kv1.3/Kv1.5 ratio in BPH VSMC. Consistent with this, arterial injury in BPH mice induced a higher degree of luminal stenosis, (84±4 % vs. 70±5 % in BPN, p<0.01), although no differences in migration and proliferation rate were observed in cultured VSMCs. The in vivo proliferative lesions were significantly decreased upon PAP-1 systemic infusion (18± 6 % vs. 58±20 % with vehicle, p<0.05). Conclusions: Hypertension leads to a higher degree of luminal stenosis in our arterial injury model, that correlates with a decreased expression of Kv1.5 channels. Kv1.3 blockers decreased in vitro VSMCs proliferation, migration, and in vivo intimal hyperplasia formation, pointing to Kv1.3 channels as promising therapeutical targets against restenosis.La versión original del artículo contiene un error. El gráfico de la página 505 es incorrecto. La corrección del mismo se encuentra en el segundo fichero "Erratum to: K+ Channels Expression in Hypertension After Arterial Injury, and Effect of Selective Kv1.3 Blockade with PAP-1 on Intimal Hyperplasia Formation".Ministerio de Economía, Industria y Competitividad (project RD12/0042/0006)Fondo de Investigación en Salud - Instituto Carlos III (project PI11/00225)VALTEC 09-1-0042Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898)Junta de Castilla y León (grant VA094A11-2

Kv1.3 channels can modulate cell proliferation during phenotypic switch by an ion-flux independent mechanism

Producción CientíficaObjective: Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. Methods and Results: Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by “poreless” mutants but disappeared when voltagedependence of gating was suppressed. Conclusion: These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally, the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target to control unwanted vascular remodeling.Instituto de Salud Carlos III (grant R006/009)Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898)Junta de Castilla y León (grant VA094A11-2